ABSTRACT The objective of this application is to first develop and validate microvessel measurement for the entire brain to enhance detection sensitivity of microvessels by ten-fold using superparamagnetic iron oxide (SPIO) enhanced susceptibility weighted imaging (SWI, SPIO-SWI) and then to investigate the interaction between glymphatic and vascular systems for waste clearance in the diabetic brain. Emerging data indicate that the glymphatic system in the brain mediates the cerebrospinal fluid (CSF)-interstitial (ISF) exchange and solute clearance from the brain parenchyma and plays an important role in neurological diseases1-6. Despite many milestone achievements, conclusive findings on the solute efflux pathways are relatively limited. Consequently, the interaction between vascular and glymphatic systems on waste clearance, especially with neurological diseases, is unclear. The paucity of research into the efflux pathway may be attributed in part to technical difficulties, such as the challenging need to perform minimally invasive in-vivo, ultra-high detection sensitivity for tube-shaped influx and efflux pathways, and whole brain imaging. Although MRI can overcome the weak points of two-photon confocal microscopy to provide non-invasive whole brain in-vivo imaging of the glymphatic system, conventional MRI sensitivity is insufficient for the required spatial resolution for investigating microvessels of glymphatic and vascular systems. We have developed highly sensitive MRI methods (Fig. 1) which significantly improve the detection sensitivity of small vessels by using the combination of high susceptibility of MRI agents with blooming effects7-9. The new methods provide excellent tools for investigating the efflux pathways of waste clearance under normal and pathophysiological conditions. Three efflux routes have been recently proposed and solutes in the brain could reach the lymphatic network by the olfactory bulb across the ethmoid plate10, 11 or by functioning conventional lymphatic vasculature in the meninges12. We found that tracer concentration in the venous system significantly increased with diabetes (Fig. 9), thus adding a new route for brain waste clearance. Based on our novel preliminary data and published studies by others, we hypothesize that, the newly developed SPIO-SWI technique significantly increases detecting sensitivity of microvessels in both vascular and glymphatic systems, and the efflux pathways of waste clearance with and without diabetes can be identified and investigated using this optimized SPIO-SWI method. To test these hypotheses, we will first (Aim 1) further develop, optimize and validate SPIO-SWI techniques to enhance the detection sensitivity for both vascular and glymphatic microvessels. We will perform computer simulation, optimize SWI technique and experimental conditions in animal studies and then validate USPIO-SWI technique by LSCM measurements. We will then (Aim 2) investigate the interaction between vascular and glymphatic systems for waste clearance in diabetic brain using the optimized USPIO-SWI technique. Data generated from this application will provide new insights into the efflux pathways between glymphatic and vascular systems in diabetic brain.